The advent of genetic engineering holds many promises. The transformation of crop plants with new transgenes encoding desirable traits promises improved crop yields and qualities. The ability to utilize plants as platforms for the bioproduction of materials (e.g., a multiplicity of monomers and polymers currently only available by chemical synthetic means) promises processes with lower production costs, higher qualities, and reduced environmental impact. The transformation of animals with new transgenes encoding desirable traits promises novel research or commercial applications, such as improved disease resistance. However, despite the tantalizing promises of genetic engineering, scientists must overcome two significant, yet related, challenges. First, it is necessary to develop techniques that overcome the consequences of gene silencing, whereby expression of the introduced foreign gene is dramatically suppressed; and second, a mechanism for silencing undesirable endogenous genes must be discovered. Both of these requirements can be achieved by conditional control of gene silencing.
Gene silencing (GS) was first recognized when the following inverse correlation was noted: multiple copies of a transgene were often associated with dramatic inhibition of that transgene's product and any host genes that possessed homology to that transgene. Now, gene silencing is known to occur by a decrease in the steady state levels of mRNA for a specific target gene, where the target gene is an endogenous gene or a transgene and is of chromosomal or viral origin.
Gene silencing is believed to have evolved as a host defense mechanism against foreign DNA elements and viruses, as it appears to be conserved in all eukaryotes and, at least, in one single cell alga. Specifically, recent studies have shown that post-transcriptional gene silencing (PTGS) in plants, RNA interference (RNAi) in worms, flies, and mammals, and quelling in fungi share common steps in gene silencing (Fire, A. Trends Genet. 15(9):358-63 (1999); Sharp, P. A. Genes Dev., 13:139-141 (1999); Sharp, P. A. and P. D. Zamore, Science, 287(5462): 2431-3 (2000)). In each form of gene silencing, double-stranded RNA (ds RNA) is an efficient trigger for the sequence-specific degradation of its cognate mRNA. Transcription of the promoter resulting in ds RNA can also lead to transcriptional gene silencing (TGS), a second form of gene silencing in plants.
Considerable efforts have focused on understanding the gene silencing phenomenon and mechanism of action in plants. A variety of review articles discuss TGS and PTGS, such as those of Waterhouse, P. M., et al. (Nature 411:834-842 (14 Jun. 2001)), Vaucheret, H. and M. Fagard (TRENDS in Genetics 17(1):29-35 (2001)), and Okamoto, H. and H. Hirochika (TRENDS in Plant Sci. 6(11):527-534 (2001)). The following summary of gene silencing is presented only as an overview.
In plants, gene silencing can be triggered by transgene duplication events (tandem repeat transgene sequences, inverted repeat transgene sequences, or multiple insertions into the chromosome) or when a sequence homologous to the target gene sequence is carried either by an infecting plant virus or by the T-DNA of an infiltrating Agrobacterium. Thus, the ds RNA trigger for TGS or PTGS can be either synthesized in vitro and then introduced into a cell; or, it can be made by transcription from a transgene construct in vivo.
Currently, TGS is not as well understood as PTGS. TGS occurs when transcription of the target gene is blocked. The process appears to be meiotically heritable and correlates with DNA template modification manifested by hyper-methylation of promoters of silenced genes or with local changes of chromatin structure. It has been postulated that TGS is a defense system against invasive DNA, such as transposable elements; but, experimental evidence for this hypothesis has not been obtained as of yet.
In contrast, post-transcriptional gene silencing (PTGS) occurs in plants when the target gene is transcribed, but its mRNA is sequence-specifically degraded before it can be translated. Thus, this reduction in mRNA is caused by an increased turnover of target RNA species, in which the transcription level of the corresponding genes remains unaffected. Corresponding gene products are therefore only able to accumulate at very low levels. One specific type of PTGS occurs as virus-induced gene silencing (VIGS), in which the sequence homologous to the target gene sequence is carried by an infecting cytoplasmically replicating plant virus. PTGS can be initiated in a variety of ways, and is thought to underlie the phenomena of co-suppression of endogenous plant genes and depressed expression of transgenes.
One of the most intriguing features of PTGS in transgenic plants is that it operates in a non-cell autonomous manner. A signal of gene silencing can move between cells through plasmodesmata and over long distances through the vascular system, directing sequence-specific degradation of target RNAs. The nature of the signal is unknown; but, based on the specificity of its degradation, it is thought to be mediated by short RNA species corresponding to the target RNA that accumulates in tissues exhibiting PTGS (i.e., RNA approximately 21-25 nucleotides in length). Furthermore, although the exact mechanism by which PTGS operates is yet to be elucidated, it has been discovered that viruses can both initiate and be a target of PTGS. A virus-induced silencing signal could therefore migrate cell-to-cell in advance of the infection front and be transported over long distances through the phloem. The effect of this intercellular signalling would be to potentiate RNA sequence-specific virus resistance in non-infected tissues and, consequently, to delay spread of the virus through the plant.
Evidence of plant viruses having evolved as a counter-defense to PTGS was determined by studies of synergistic viral disease, where co-infection with two heterologous viruses led to much more severe symptoms than did infection with either virus alone. Transgenic plants expressing the 5′ proximal region of the tobacco etch potyviral (TEV) genome (i.e., PI/HC-Pro sequence) developed synergistic disease when infected with any of a broad range of plant viruses (Pruss, G., et al. Plant Cell 9:859-868 (1997)). This suggested that expression of the PI/HC-Pro sequence interfered with a general antiviral system in plants that permitted viruses to accumulate beyond the normal host-mediated limits. More recently, a plant viral protein (HC-Pro) has been identified that interferes with the induction of PTGS (Anandalakshmi, R. et al. Proc. Natl. Acad. Sci. USA., 95(22): 13079-84 (1998); Brigneti, G. et al. EMBO J., 17(22): 6739-46 (1998); Kasschau, K. D. and J. C. Carrington, Cell, 95(4): 461-70 (1998)), further supporting the idea that PTGS may be linked to natural antiviral resistance systems in plants. It was shown that this synergism was due to suppression of a host defense mechanism by the Hc-protease (HcPro) encoded in the potyviral genome (e.g., tobacco vein mottling virus, tobacco etch virus, and potato virus Y). Subsequent studies further established that HcPro was a suppressor of PTGS and provided a link between PTGS and antiviral defense. Thus, suppressors of gene silencing can be used inter alia for improving expression of desirable genes, particularly heterologous genes, in plants (WO 98/44097).
Despite the previous work identifying suppressors of gene silencing, these solutions are limited by the undesirable side effects of many, if not all, suppressors of gene silencing or by mutations in host gene silencing. A significant contribution to the art, therefore, would be the development of a general mechanism whereby conditional manipulation of PTGS or other gene silencing pathways would serve as an effective means for modulating expression of endogenous or foreign target genes. That is, it would be desirable to control when—and in which tissue—the expression of the target sequence is enhanced, decreased, or silenced.
Currently, few methods exist that provide for the conditional manipulation of the gene silencing pathways to silence 1.) specific gene-silencing genes, thereby permitting higher level expression of transgenes that would ordinarily be silenced; and, 2.) undesirable endogenous genes, thereby permitting their down-regulation. Transgenes encoding hairpin or ds RNA have been used to silence target genes (Smith et. al. Nature 407:319 (2000); De Buck et. al. Plant Mol. Biol. 46:433 (2001)). And, silencing of a gene-silencing gene (i.e., the DICER gene) has also been demonstrated in the art (Hutvagner et. al. Science 293:834 (2001)). However, it has not been shown that such silencing can be conditionally activated. Conditional silencing is essential, as constitutive silencing is not desirable when silencing of a target gene is lethal or deleterious to normal growth and development of an organism. Additionally, conditional silencing of a gene involved in gene silencing would permit production of high levels of materials in transgenic plants that normally are susceptible to gene silencing. And further, methods of conditionally silencing the expression of an endogenous or foreign target gene would enable economic production of desired chemicals, monomers, and polymers, with high levels of expression being restricted to transgenic biomass (production tissue) either just prior to, or after, its harvest for extracting the desired product. Thus, regulated ds RNA expression would be desirable for regulated gene silencing of target genes at a specific developmental time or in a specific tissue or generation.
Site-specific recombination [Odell et al., Plant Physiol. 106:447-458 (1994); Odell et al., WO 91/09957 (1991); Surin et al WO 97/37012 (1997); Ow et al., WO 93/01283 A1 (1992); Russel et al., Mol. Gen. Genet. 234:49-59 (1992); and Hodges et al., U.S. Pat. No. 6,110,736] in plants has been demonstrated. Furthermore, regulated site-specific recombination (SSR) in plants has also been demonstrated in plants using binary expression systems, where one transgenic cassette carries a site-specific recombinase and the other carries the inactive trait. The expression of this trait gene (TG) is blocked by the presence of a ‘blocking’ or ‘STOP’ fragment, flanked by the cognate SSR sites, which blocks transcription and/or translation of the TG. Recombinase expression leads to SSR and removal of the “blocking” DNA fragment, thereby permitting transgene activation. This can be regulated to occur in a specific developmental stage of the plant, tissue, or generation (Yadav et al., WO 01/36595 A2; WO 00/17365 A2; U.S. Pat. No. 6,077,992; EP1115870 A2). However, a sitespecific recombination system has never been used as a method to control gene silencing. Thus, the problem to be solved, therefore, is to develop a system for conditionally regulating gene silencing through the implementation of SSR systems, such that target gene expression leads to production of ds RNA which thereby triggers gene silencing.
Applicant has solved the stated problem in the present invention through the development of a method for conditional regulation of gene silencing based on site-specific recombinase systems. These systems comprise a recombinase element (which leads to expression of an active recombinase enzyme) and a gene silencing-recombinase element (which leads to expression of the target sequence as ds RNA).